The chromatographic separation of polar compounds is associated with a variety of difficulties. Reversed-phase (RP) columns are widely used for separating molecules via hydrophobic interaction. However, conventional alkyl-bonded reversed-phase media (e.g., C18 and C8) are often not suitable for retaining or separating highly polar molecules (Neue, “HPLC Columns—Theory, Technology, and Practice”, WILEY-VCH, New York, 1997, 183-203). Likewise, ion-pairing chromatography is often problematic because of long equilibration times and incompatibility with mass spectroscopy (MS).
Normal-phase chromatography can also be used to separate polar molecules. A wide range of solvents can be utilized to adjust the selectivity of a particular separation (Neue, “HPLC Columns—Theory, Technology, and Practice,” WILEY-VCH, New York, 1997, 164-182). However, due to the sensitivity of normal-phase media to the presence of small concentrations of polar contaminants in the mobile phase, lengthy equilibrating times are common and/or reproducibility problems may occur. Moreover, normal-phase chromatography is often inapplicable due to poor solubility of highly polar compounds in organic solvents.
HILIC (HydrophILic Interaction Chromatography or Hydrophilic Interaction LIquid Chromatography) is a form of normal phase liquid chromatography. HILIC fills the gap between reversed-phase and normal phase chromatography. Appropriate HILIC phases can retain polar compounds while using organic mobile phases, which incorporate only a small percentage of water or an aqueous buffer, typically between about 5 and 30% (Neue, “HPLC Columns—Theory, Technology, and Practice,” WILEY-VCH, New York, 1997, 217-223).
In HILIC, retention increases with the polarity of the analyte and decreases with the polarity of the mobile phase. One possible retention mechanism of HILIC is the partitioning of the analytes between a water-rich stationary phase and a water-poor mobile phase (A. J. Alpert, J. Chromatogr. 1990, 499, 117-196). Additional benefits of HILIC include complementary selectivity to reversed-phase columns, enhanced sensitivity in mass spectrometry, and simplified sample preparation procedures. Consequently, HILIC provides a mechanism for the separation and analysis of a wide range of analytes, such as carbohydrates, amino acids, peptides, oligonucleotides and phospholipids, but also small-molecule drugs and their metabolites.
In general, the packing materials for normal phase chromatography and HILIC can be grouped into the following five categories:
(1) Simple, unbonded silica (see e.g., B. A. Olsen, J. Chromatogr. 2001, A 913, 113-122);
(2) Neutral silica-based packings bearing diol groups (H. Tanaka, X. Zhou, and O. Masayoshi, J. Chromatogr. 2003, A 987, 119-125);
(3) Silica-based packings with amide functionality (A. J. Alpert, J. Chromatogr. 1990, 499, 117-196);
(4) Silica-based packings bearing anion-exchange or cation-exchange functionalities (Neue, “HPLC Columns—Theory, Technology, and Practice,” WILEY-VCH, New York, 1997, 217-223); and
(5) Zwitterionic silica-based packings, such as those currently offered by Sequant.
Unbonded silica was one of the first and most extensively used chromatographic packing material because it was readily available. As discussed above, this material has several drawbacks including poor reproducibility and long equilibration times as well as a limited operating pH range. To overcome these difficulties, silica gels covalently modified with a diol moiety (diol phase) were developed. In these diol phases, silanol groups are functionalized with hydroxyalkyl groups. The resulting stationary phases offer improved reproducibility, shorter equilibration times and a wider operating pH range. In addition, diol-phases are often characterized by reduced secondary interaction compared to its ionizable counterparts.
However, these and other conventional HILIC materials have inadequate hydrophobicity, making them unsuitable for separating molecules under RP conditions. Therefore, it is highly desirable to develop stationary phases that combine both HILIC and RP characteristics. The present invention addresses these and other needs.